Authors :
Presenting Author: Durgesh Tiwari, PhD – Cincinnati Childrens Hosp Medical Center
Maggie Sheridan, Ms – Neurology – Cincinnati Childrens Hosp Medical Center; Rachana Kode, . – Neurology – Cincinnati Childrens Hosp Medical Center; Rishav Mukherjee, Mr – Neurology – Cincinnati Childrens Hosp Medical Center; Andrew Snider, Mr – Neurology – University of Cincinnati, Cincinnati, Ohio 45229, USA; Steve Danzer, PhD – Professor, Anesthesia, Cincinnati Childrens Hosp Medical Center; Christina Gross, PhD – Associate Professor, Neurology, Cincinnati Childrens Hosp Medical Center
Rationale:
Mutations in Contactin-associated protein like 2 (CNTNAP2) have been linked to autism and epilepsy. Cntnap2 KO mice display social behavioral deficits at early ages and later develop spontaneous seizures and show selective impairment of inhibitory neuronal transmission in the hippocampal CA1 region. The mechanisms of how Cntnap2 deletion leads to altered inhibition is unknown. MicroRNAs (miRNAs) regulate post-transcriptional gene expression and are crucial regulators of genes involved in epilepsy. MiR-324-5p inhibition was shown to provide neuroprotection in mouse models of epilepsy and miR-218-5p is proconvulsive in mouse models of acquired epilepsy. Manipulating these candidate microRNAs in Cntnap2 KO mice could help identify Cntnap2-mediated mechanisms regulating neuronal inhibition and seizure development and may serve as therapeutic targets. Here, we study the effect of antisense-mediated inhibition of miR-324-5p and miR-218-5p on seizure susceptibility, interictal spike activity and hippocampal inhibitory neuronal networks
in adult and older Cntnap2 KO mice.Methods:
Cntnap2 KO and littermate control mice were implanted with cortical EEG electrodes at adult (four to six months) and older (twelve to sixteen months) age points to confirm spikes and seizure occurrence. A different cohort of adult and older mice were implanted with electrodes and ICV injected with miR-324-5p, miR-218-5p antagomir and scrambled (SCR) control to assess seizure susceptibility to kainic acid (KA) (adult mice) and frequency of spontaneous seizures (older mice), respectively. Later, mouse brain tissues were collected for immunohistological analyses and mRNA target analyses.
Results:
EEG analysis in younger mice showed increased spike activity but no spontaneous or handling-induced seizures. Older mice displayed both spikes and spontaneous and handling-induced seizures. EEG analyses in younger mice showed significant delayed seizure onset after kainic acid in the miR-324-5p antagomir treatment group and reduced latency to seizure onset post miR-218 treatment (n=7-9, one-way ANOVA, p=0.012). Older mice on average showed increased numbers of seizures (non-significant) in miR-218 antagomir-injected mice compared with 324-5p or scrambled control (one-way ANOVA, p=0.134). No difference in the average spike count, EEG power analysis or hippocampal morphology was observed among the treatment groups (p > 0.05). MiR-324-5p inhibition showed an increase in CA1 inhibitory interneurons in four to six month-old mice (p=0.002).
Conclusions:
MiR-324-5p antagomir treatment confers neuroprotection to both seizing and non-seizing networks in a Cntnap2 KO mouse model of epilepsy, whereas miR-218 antagomirs did not have significant effects. These findings illustrate the role of individual miRNAs inhibition and inhibition strategies in regulating seizures severity in Cntnap2 KO mice. Ongoing work also investigates the role of candidate microRNA inhibition in induced (DFP) mouse model of epilepsy.
Funding:
This research was supported by a postdoctoral fellowship from the American Epilepsy Society (D.T), CCTST-MTRS (D.T), and NIH grant R01NS092705 (C.G.), R01NS107453 (C.G).